JULIA MOURÃO BRAGA DINIZ
EFEITO DO MTA FILLAPEX SOBRE A ATIVIDADE
DE MACRÓFAGOS PERITONEAIS
FACULDADE DE ODONTOLOGIA
UNIVERSIDADE FEDERAL DE MINAS GERAIS
BELO HORIZONTE
JULIA MOURÃO BRAGA DINIZ
EFEITO DO MTA FILLAPEX SOBRE A ATIVIDADE
DE MACRÓFAGOS PERITONEAIS
FACULDADE DE ODONTOLOGIA
UNIVERSIDADE FEDERAL DE MINAS GERAIS
BELO HORIZONTE
2013
Dissertação apresentada ao Colegiado do programa de Pós-Graduação da Faculdade de Odontologia da Universidade Federal de
Minas Gerais, como requisito parcial para a obtenção do grau de Mestre em Odontologia - área de
concentração em Endodontia
FICHA CATALOGRÁFICA
Elaborada pela Biblioteca da Faculdade de Odontologia - UFMG D585a Diniz, Júlia Mourão Braga.
2013 Efeito do MTA Fillapex sobre a atividade de macrófagos T peritoneais / Júlia Mourão Braga Diniz. – 2013.
88 f. : il.
Orientador: Antônio Paulino Ribeiro Sobrinho. Co-orientadora: Leda Quércia Vieira.
Dissertação (Mestrado) – Universidade Federal de Gerais, as Faculdade de Odontologia.
1. Macrófagos peritoneais. 2. Materiais restauradores do canal radicular – Análise. 3. Teste de materiais. I. Ribeiro Sobrinho, Antônio Paulino. II. Vieira, Leda Quércia. III. Universidade Federal de
Minas Gerais. Faculdade de Odontologia.IV Título.
DEDICATÓRIA
Ao meu marido, Henrique, por toda ajuda e pelo apoio incondicional. Com
você ao meu lado, todas as dificuldades se tornam menores e a vida é sempre mais
gostosa. Obrigada por entender meus momentos de ausência.
Aos meus pais, Fernando e Isa, meus primeiros mestres. Foi o apoio e amor
incondicional de vocês que me guiaram até aqui. Não existem palavras para explicar a
importância de vocês na minha vida.Obrigada por tudo!!
Aos meus irmãos, Daniel e Fernanda, amigos e companheiros de uma vida
inteira. Obrigada por estarem sempre ao meu lado, torcendo pelo meu sucesso.
Aos meus amigos, por fazerem a vida mais alegre e gostosa.
AGRADECIMENTOS ESPECIAIS
Ao Professor Doutor Antônio Paulino Ribeiro Sobrinho, pela sabedoria, paciência,
compreensão, estímulo constante e orientação sem fim. Sua presença nesta etapa da minha
vida foi fundamental.Obrigada por tudo!
À professora Doutora Leda Quercia Vieira, por sua atenção, paciência e
disponibilidade em todas as etapas desta caminhada.
Aos colegas Graziele e Waldionê pela paciência e disponibilidade. A ajuda de vocês
foi preciosa!!!
Ao Ricardo por guiar meus primeiros passos e por toda ajuda durante a realização
desse trabalho.
À Kamilla por toda ajuda e, principalmente, pela amizade construída ao longo
dessa caminhada.
AGRADECIMENTOS
Aos meus colegas do laboratório de Gnotobiologia e Imunologia
(ICB-UFMG):Graziele, Caio, Leonardo, Waldionê, Paula, Matheus, Liliane, Peter, Brenda,
Mateus e Clara pela parceria nos trabalhos desta pesquisa.
Aos colegas de pós graduação, pela parceria e amizade. Em especial, aos queridos
amigos: Andréia, Giovani, Marcela e Wilson pelos momentos de risos e alegria.
À professora Renata Martins por toda ajuda na conclusão desse trabalho.
Á Taia pela ajuda e pela disponibilidade.
Ao Professores do mestrado, que tanto contribuíram para minha formação pessoal
e profissional. Em especial, à Professora Guiomar .
RESUMO
A obturação do sistema de canais radiculares após a limpeza e modelagem é fundamental para o sucesso do tratamento endodôntico. Os materiais obturadores devem ter propriedades físicas e químicas favoráveis. Além disso, é altamente desejável que sejam biocompatíveis, porque, muitas vezes, são colocados em contacto íntimo com os tecidos periapicais, através do forame e das comunicações acessórias apicais. O Agregado Trióxido Mineral (MTA) é conhecido por sua excelente biocompatibilidade e capacidade de vedação. No entanto, apesar das suas características favoráveis, o MTA não apresenta as propriedades físicas necessárias para ser usado como cimento obturador. Em uma tentativa de combinar as propriedades físico-químicas de um cimento endodôntico com as propriedades biológicas do MTA, um cimento obturador à base de MTA (MTA Fillapex ®-Angelus, Londrina, Paraná, Brasil), foi introduzido no mercado. Neste estudo, testou-se o efeito do MTA e do MTA Fillapex (FLPX) sobre a atividade de macrófagos inflamatórios peritoneais M1 (provenientes de camundongos C57BL/6) e M2 (provenientes de camundongos Balb/c). Foram avaliadas: a viabilidade e aderência celular, a fagocitose de Saccharomyces Boulardii, a produção de ROIs, quando os macrófagos foram ou não estimulados com zymosan, e a produção de TNF, IL-12, IL-10 e NO, quando estimulados ou não com o Fusobacterium nucleatum,
pelos M1 e M2. Os macrófagos M2 não produziram IL-12. O FLPX diminuiu a expressão de TNF-α por macrófagos M2 estimulados com F.nucleatum. Podemos
concluir que o MTA Fillapex inibiu atividades importantes dos macrófagos peritoneais M1 e M2.
ABSTRACT
Root canal filling after cleaning and shaping is paramount for a succesful endodontic treatment. Filling materials should have favorable physical and chemical properties. Furthermore, is highly desirable for them to be biocompatible, because, often, they are in close contact with periapical tissues through foramen and accessory periapical communications. The Mineral Trioxide Aggregate (MTA) is known by its excellent biocompatibility and sealing aptitude. Although its favorable characteristics, MTA do not have the physical properties needed to be used as sealing cement. In a attempt to combine endodontic sealers physical and chemical properties with MTA biological properties, a filling cement based on MTA (MTA Fillapex® - Angelus, Londrina, PR, Brazil), was introduced in the market. In this study, MTA and MTA Fillapex (FLPX) effect were tested over M1 (C57BL/6 mice provided) and M2 (Balb/c mice provided) inflammatory macrophages. Cellular viability, cellular adherence, phagocytosis of Saccharomyces Boulardii, production of reactive oxygen intermediates (ROIs), nitric oxide (NO), TNF, IL-12 and IL-10 were assessed . Production of ROIs was stimulated by zymozan. Production of cytokines was stimulated with
Fusobacterium nucleatum, Peptostreptococcus anaerobius and IFN-ɣ. FLPX impaired
LISTA DE ABREVIATURAS E SIGLAS
1. ELISA: Enzime-linked immunoabsorbent assay 2. FLPX: MTA Fillapex
3. IFN: interferon 4. IL: interleucina
5. LPS: lipopolissacáride 6. M1: macrófago do tipo 1 7. M2: macrófago do tipo 2
8. MTA: mineral trioxide aggregate 9. NO: óxido nítrico
SUMÁRIO
1. Introdução e relevância...16
2. Objetivos...19
3. Trabalhos científicos...21
4. Conclusão...76
16
17
1 Introdução e Relevância
O tratamento endodôntico consiste na limpeza mecânico-química e formatação do sistema de canais radiculares, seguidos por uma obturação hermética (Brkanic et al., 2005). A obturação do sistema de canais radiculares (SRC) é considerada uma das etapas de importância singular dentro da terapia endodôntica. Tem como objetivo vedar tridimensionalmente o SRC, impedindo a percolação de fluidos dos tecidos perirradiculares e a contaminação pelos microrganismos presentes na cavidade oral. A obturação deve impedir, também, que qualquer microrganismo que não tenha sido removido durante os procedimentos de limpeza e formatação recolonize os canais radiculares, além de criar um ambiente biológico favorável para que se processe a cicatrização dos tecidos periapicais (Nguyen, 1994).
Tradicionalmente, para a confecção da obturação dos sistemas de canais radiculares utilizam-se materiais semi-sólidos, como a gutta percha, em combinação com cimentos ou pastas obturadoras. Estes devem apresentar propriedades físico-químicas favoráveis (Grossman, 1988), desejando-se também que apresentem biocompatibilidade, por serem colocados em íntimo contato com os tecidos periapicais via forame apical, ramificações e canais acessórios (Bernath & Szabo 2003). Em contato com os tecidos, os cimentos obturadores podem desencadear processos inflamatórios de diversas intensidades, afetando o processo de reparo (Langeland 1974, Giardino et al. 2006, Gomes-Filho et al. 2008, Batur & Erserv 2008).
18 imediatamente incorporado pela comunidade odontológica. Desde então, o MTA vem sendo largamente utilizado na terapia endodôntica devido a sua biocompatibilidade e excelente capacidade de selamento (Torabinejad & Chivian 1999, Rezende et al. 2007, Yasuda et al. 2008, Scarparo et al. 2010, Parirokh & Torabinejad 2010, Batur et al 2013, Guven et al. 2013, Hirschberg et al. 2013).
O MTA é composto de partículas de silicato tricálcico hidrofílicas, aluminato tricálcico, óxido tricálcico, óxido de silicato e outros óxidos minerais (Lee et al. 1993, Torabinejad et al. 1993, Torabinejad & Chivian 1999). O MTA foi inicialmente indicado como material retrobturador nas cirurgias periapicais e no selamento de comunicações entre o sistema de canal radicular e os tecidos adjacentes. O MTA quando aplicado junto ao osso permite a deposição de cemento e a formação óssea, facilitando a regeneração do ligamento periodontal (Schwartz et al. 1999). Atualmente, vem sendo utilizado na indução da apexificação em dentes com rizogênese incompleta e, também, no capeamento pulpar direto (Saidon et al. 2003). Entretanto, apesar de suas propriedades desejáveis, que o credencia como o material de escolha em vários procedimentos endodônticos, o MTA não apresenta propriedades compatíveis para ser utilizado como cimento endodôntico. Dentre elas, destacam-se: a dificuldade de manipulação, reduzido tempo de presa e de trabalho (Roberts et al. 2008).
Várias composições de cimentos endodônticos foram desenvolvidas com o objetivo de ser biocompatível aos tecidos periapicais, respeitando-se suas propriedades físico-químicas (Al-Hiyasat et al., 2010). Neste contexto surgiu o MTA Fillapex®-(Angelus; Londrina, Paraná, Brazil), na tentativa de combinar as propriedades físico-químicas de um cimento endodôntico com as propriedades biológicas do MTA.
19 fabricante, o MTA fillapex, que possui 13% de MTA, apresenta como características: ser biocompatável, apresentar efeitos antimicrobianos, induzir o reparo biológico graças à liberação de íons cálcio, promover selamento adequado, e radiopacidade elevada em virtude da presença do óxido de bismuto (Nagas et al.2012, Assmann et al. 2012).
Nos tecidos perirradiculares inflamados, células imunocompetentes estarão presentes, havendo um predomínio de macrófagos (Van Dyke 2008). Os macrófagos desempenham um importante papel na patogênese do processo inflamatório, constituindo uma das primeiras células que entram em contato com partículas estranhas. Desempenham um papel regulatório direcionando o processo inflamatório (Hasturk et al.2012).
As principais funções dos macrófagos são: eliminar antígenos invasores, recrutar células de defesa para o sítio da infecção, sintetizar citocinas e quimiocinas, promover o
clearance de neutrófilos e ativar a resposta imune adaptativa mediada por linfócitos
(Hasturk et al.2012). Os macrófagos inflamatórios diferem dos macrófagos residentes especialmente pelo aumento de sua capacidade fagocítica e pela habilidade aumentada de gerar metabólitos tóxicos de oxigênio e nitrogênio (Fujiwara & Kobayashi, 2005). As citocinas e produtos microbianos afetam diferentemente a função destas células ativando ou desativando-as (Mantovani et al., 2004).
20 ROI são produtos de redução intermediária do oxigênio, como o superóxido, peróxido de hidrogênio, e radicais hidroxíla, bem como os produtos reativos dos mesmos(Nathan & Shiloh 2000). Por outro lado, os macrófagos M2 convertem, preferivelmente, a arginina em uréia e ornitina, o que resulta na produção de colágeno e proliferação celular.
A polarização M1/M2 é diferenciada pelos níveis constitutivos de IL-12/IL-10 (Bastos et al.,2002; Mosser, 2003; Mantovani et al.,2004). Macrófagos M1 produzem tipicamente altas concentrações de IL-12 e baixas de IL-10, enquanto macrófagos M2 secretam altas concentrações de IL-10 e baixas de IL-12 (Verreck, et al., 2004; Edwards
et al., 2006).
Tendo em vista que a biocompatibilidade é uma propriedade biológica desejável a um material destinado ao uso biológico, alguns estudos avaliaram a citotoxicidade do MTA Fillapex. Bin et al. (2012), Yoshino et al. (2013), e Silva et al. (2013) estudaram o efeito do MTA Fillapex sobre a viabilidade celular de fibroblastos. Estes autores demonstraram que o MTA Fillapex reduziu significativamente a viabilidade dessas células. Salles et al. (2012) também observaram uma redução na viabilidade de osteoblastos após a exposição a diferentes cimentos endodônticos, dentre eles o FLPX.
Zmener et al. (2012) e Tavares et al. (2013) analizaram a resposta do tecido subcutâneo de ratos à cimentos endodônticos. Zmener et al. (2012) concluiram que o FLPX não apresentou nenhuma vantagem em relação à biocompatibilidade tecidual quando comparado ao cimento de Grossman. Tavares et al. (2013) observaram uma maior ocorrência de abscessos nos grupos tratados com o FLPX e Endofill quando comparado ao AH Plus e ao grupo controle não tratado.
21 reduziam no número de unidades formadoras de colônia em biofilmes induzidos em dentina bovina. Estes cimentos também apresentaram uma maior solubilidade quando comparados ao AH Plus, Sealer 26, Epiphany SE, Activ GP e a um cimento à base de MTA.
Tendo em vista que a adesão é uma propriedade físico-química desejável à um cimento endodôntico, Sagsen et al. (2011) compararam a adesividade às paredes do canal promovida pelo FLPX àquela de outros materiais obturadores. O FLPX apresentou menor capacidade de adesão quando comparado ao I Root SP e ao AH Plus.
As citocinas são importantes mediadores envolvidos na patogênese da reabsorção óssea e do processo inflamatório. Gomes-Filho et al. (2009) investigaram os efeitos de um cimento endodôntico a base de MTA, o Endo CPM sealer (CPM Sealer; EGEO SRL, Buenos Aires, Argentina), do Sealapex e do MTA Ângelus sobre a expressão da IL1-β e a IL-6 em culturas de fibroblastos. Observaram que nenhum dos cimentos testados alterou os índices de viabilidade celular e que todos aumentaram a expressão da IL-6. A expressão da IL-1β foi maior no grupo tratado pelo MTA. Não se observou diferença estatística entre os outros grupos experimentais em relação ao grupo controle.
22
23
2. Objetivos
Objetivo geral:
Avaliar o efeito dos cimentos MTA fillapex (Ângelus) e do MTA (Ângelus) nas respostas dos macrófagos M1 e M2 murinos in vitro.
Objetivos específicos:
Avaliar a viabilidade celular de macrófagos M1 e M2, em contato com o MTA Fillapex (Ângelus) e com o MTA (Ângelus).
Avaliar a capacidade de aderência dos macrófagos M1 e M2, após contato com o MTA Fillapex (Ângelus) e com o MTA (Ângelus).
Avaliar a capacidade de fagocitose dos macrófagos M1 e M2, após contato com MTA Fillapex (Ângelus) e com o MTA (Ângelus).
24
25
Assessment of the cytotoxicity of a mineral trioxide aggregate-based
sealer with respect to macrophage activity.
Julia Mourão Braga1, Ricardo Reis Oliveira1, Renata de Castro Martins2, Leda Quércia
Vieira3,4, Antonio Paulino Ribeiro Sobrinho1.
1Departamento de Odontologia Restauradora, Faculdade de Odontologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil;
2 Departamento de Odontologia Social e Preventiva, Faculdade de Odontologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil;
3Departamento de Bioquímica e Imunologia Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil;
26 Abstract
Aim
To assess the influence of co-culture with mineral trioxide aggregate (MTA) and MTA Fillapex (FLPX) on the viability, adherence and phagocytosis activity of M1 and M2 peritoneal macrophages.
Methodology
Cellular viability, adherence and phagocytosis of Saccharomyces boulardii were assayed in the presence of capillaries containing MTA and MTA Fillapex. The data
were analyzed using parametric (Student’s t) and nonparametric (Mann–Whitney) tests. Results
FLPX was severely cytotoxic and decreased cell viability, adherence and phagocytic activity of both macrophage subtypes. Cells that were treated with MTA Fillapex remained viable ( 80%) for only 4 hours after stimulation. M1 macrophages presented higher adherence ability and higher phagocytic activity compared with M2 macrophages.
Conclusion
FLPX was severely cytotoxic and remarkably decreased M1 and M2 macrophage viability. FLPX also impaired cell adhesion properties and the phagocytic ability of both macrophage subtypes. Conversely, MTA did not interfere with the viability, adherence or phagocytic activity of M1 and M2 macrophages.
27
Introduction
A complete sealing of the root canal system after cleaning and shaping is critical for a successful endodontic treatment. Root canal are traditionally filled with gutta-percha cones and a sealer. It is highly desirable for sealers to be biocompatible because they can be placed in intimate contact with periapical tissues through the apical foramen and accessory communications.1 Moreover, sealers may release substances that may generate periapical inflammatory reactions.
Mineral trioxide aggregate (MTA) has been extensively studied and widely accepted for its biocompatibility and excellent sealing capacity.2-4 In an attempt to combine the physicochemical properties of the root canal sealer with the biological properties of MTA, an MTA-based sealer (MTA Fillapex®-Angelus; Londrina, Paraná, Brazil) was introduced to the market.
Macrophages are key cells involved in inflammation during chronic or healing processes.5 According to their ability to elicit different responses, macrophages have been divided into two subtypes: M1 and M2.6-10 M1 macrophages mediate resistance to pathogens,11 while M2 macrophages are involved in healing process.6,7,9
Few studies have analyzed the cytotoxicity of MTA Fillapex, and no one has looked for its effects on macrophage viability and activity. This study assessed the influence of MTA and MTA Fillapex on viability, adherence and phagocytic activity of M1 and M2 macrophages.
28
Materials and methods
Mice
Male and female 4- to 8-week-old C57BL/6 and Balb/c mice were obtained from CEBIO (UFMG, Belo Horizonte, Brazil) and kept in a conventional animal house with barriers as well as temperature and light control. Food and water were offered ad
libitum.
Isolation of macrophages
Cells were isolated from the peritoneal cavity of C57BL/6 (M1 macrophages) and Balb/c (M2 macrophage) mice 5 days after 2 mL of 3% thioglycolate medium (Biobrás S.A.,Montes Claros, MG, Brazil) was injected into the peritoneum. The cells were resuspended in complete medium (RPMI 1640; Sigma Chemical Co., St Louis, MO, USA), supplemented with 10% of fetal calf serum (Nutricell, Campinas, SP, Brazil), 0.1% β-mercaptoethanol (0.05 mol L-1) (Sigma Chemical Co.), 0.2% penicillin (100 U mL-1)/streptomycin (0.1 mg mL-1) and 200 mmol L-1L-glutamine.12
MTA and MTA fillapex manipulation
29 Cell viability
The viability of cells was determined using two methods: the trypan blue exclusion assay and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Cell viability was assayed by trypan blue exclusion in 24-well culture plates ( 2 x 105 cells/mL for 2, 4, 6, 8, 10 and 24 hours) and in propylene tubes (1 x 106 cells/mL for 24 hours), as described previously.3,12 Briefly, cells were incubated in the presence of capillary tubes in 1 ml of RPMI (Sigma Chemical Co.) containing 10% fetal calf serum (Nutricell, Campinas, SP, Brazil), 2mM of L-glutamine, 100 U/ml of penicillin and 100 µg/ml of streptomycin at 37°C and 5%CO2 humidified atmosphere containing 5% CO2. After the incubation periods, 100 µl of 0.25% trypan blue (Sigma Chemical
Co.) in saline were added, and the cultures were examined under an inverted microscope. At least 300 cells were counted per culture, and the cultures were performed in triplicate. The results are expressed as percentage of viability. The experiment was repeated three times.
30 dissolved when removing the culture medium and adding 100 µ L of dimethyl sulfoxide solvent (Sigma-Aldrich) to each well.
The absorbance was measured at 540 nm using a microplate reader (PowerWave HT, BioTek, USA). The formazan content of each well was computed as a percentage of the control group.13 The results were expressed as the percentage viability. The experiments were repeated 3 times in triplicate.
Cell adherence
Polypropylene tubes containing macrophages (1 x 106 cells/mL) were incubated for 2 h at 37°C with capillaries (MTA, FLPX, and control groups) in a humidified atmosphere containing 5% CO2. Tubes were agitated in a vortex agitator for 15 s at low
speed. An aliquot (20 μL) of the cellular suspension was removed, placed into a Newbauer chamber and incubated for 1 h at 37°C as described above. The percentages of adherent and nonadherent macrophages were then established by counting under an optical microscope.14 Spread cells were counted as adhered cells. At least 300 cells were counted per sample.
Phagocytosis assay
31 covered for 1 min with 1 mL of 1% tannic acid (Merk) so that the distinction could be made between extracellular and intracellular yeast cells. One drop of fetal calf serum was applied onto each coverslip. The dried coverslips were stained with Panótico Rápido (Laborclin Ltd, Pinhais, PR, Brazil) and glued to glass microscope slides with Entellan (Merck). The slides were observed under an optical microscope at 1000X magnification using an oil immersion objective.15 The cells were counted until 200 macrophages with phagocytosed yeast were found. The results are expressed as percentages.
Statistical Analysis
The data were analyzed using parametric (Student’s t) and nonparametric (Mann–
Whitney) tests (P < 0.05). Analyses were performed using the Statistical Package for Social Sciences (version 18.0; SPSS Inc., Chicago, IL, USA).
Results
Cell viability
32 viability assay showed that more than 90% of the cells in the FLPX group died 24 hours after capillary exposure (data not shown). Interestingly, MTA-treated macrophages of both subtypes showed greater viability compared with control macrophages of both
subtypes (p ≤ 0.021) when cell viability was assayed by MTT (data not shown).
Cell adherence
M1 macrophages in both control and MTA groups presented higher adherence ability compared with M2 macrophages (p = 0.02) (Figure 2). However, the adherence of both macrophage subtypes was impaired by FLPX (p < 0.05) when compared with MTA and control groups.
Phagocytic activity
The capacity of mouse peritoneal cells to uptake yeast cells was assayed in this study (Figure 3).Under the control conditions, the M1 macrophages showed statistically higher phagocytic ability compared with the M2 macrophages (P = 0.033). However, both macrophage subtypes showed significant impairment in phagocytosis of S.
boulardii in the presence of FLPX (p < 0.05), and M1 cells exhibited enhanced
phagocytic activity compared with M2 cells (p < 0.05).
Discussion
Successful root canal treatment depends on the elimination of intracanal infection followed by effective and biocompatible root canal filling to avoid reinfection and irritation of the periradicular tissue.16 Thus, the biocompatibility of the endodontic root canal sealer is an important requirement for achieving this goal.17,18
33 elimination of invading bacteria, recruitment of other cells to the site of infection, clearance of the excess neutrophils, production of cytokines and chemokines and activation of the lymphocyte-mediated adaptive immune response.5 Macrophages, together with neutrophils, are responsible for phagocytosis and digestion of microorganisms and foreign substances.20
Using a well-tested methodology,3,12,21 the present study evaluated and compared the macrophage immune response to MTA Fillapex and MTA. The viability, adherence and phagocytic activity of M1 and M2 macrophages were assayed.
FLPX remarkably decreased cell viability under all conditions tested. Accordingly, other studies have reported that FLPX reduced cell survival rates. 22-24 It is important to note that FLPX contains toxic components, such as salicylate resin, which may explain its negative effects on cell viability. Previous studies have evaluated the effect of salicylate resin on a human fibrosarcoma cell line (HT-1080) and observed that it induces apoptosis.25,26
In this study, MTA promoted enhanced viability of M1 and M2 macrophages as previously described.3 MTA was also shown to exert similar effects on fibroblasts,27,28 osteoblasts 29,30 and macrophages.3,10,31 Conversely, in this study, the cells treated with FLPX remained viable (> 80%) for only 4 h (Figure 1C). This brief period of cell viability determined our next experiments with macrophage adherence and phagocytosis. In these experiments, the cells were incubated for 2 h to ensure substantial viability of macrophages treated with FLPX.
34 clearly showing that this sealer is able to impair immune system responses. Accordingly, lower cell adherence in the presence of ZOE sealers 12,21,34 and in the presence of other endodontic materials 35-37 has been shown. On the other hand, MTA did not interfere with macrophage 3, osteoblast 29 or osteosarcoma cell line adherence 33. Interestingly, when M1 and M2 control groups were compared, M1 macrophages showed significantly higher adherence compared with M2 macrophages.
Additionally, the ability of macrophages to phagocytose S. boulardii in the presence of MTA and FLPX sealers was analyzed. This yeast was selected because of its size, which makes counting easier and therefore allows greater data precision.3 The M1 macrophages showed significantly greater phagocytic ability than M2 macrophages in all tested groups. Consistent with this result, Oliveira Mendes et al. 21 have reported that M2 macrophages treated with two different zinc oxide eugenol-based materials also showed a decreased capacity for phagocytosis of S. boulardii.
35
References
1. Bernath M, Szabó J. Tissue reaction initiated by different sealers. Int Endod J 2003;36:256–61.
2. Torabinejad M, Chivian N. Clinical applications of mineral trioxide aggregate. J Endod 1999;25:197–205.
3. Rezende TM, Vieira LQ, Cardoso FP, Oliveira RR, de Oliveira Mendes ST, Jorge ML, Ribeiro Sobrinho AP. The effect of mineral trioxide aggregate on phagocytic activity and production of reactive oxygen, nitrogen species and arginase activity by M1 and M2 macrophages. Int Endod J 2007;40:603-11. 4. Scarparo RK, Haddad D, Acasigua GA, Fossati AC, Fachin EV, Grecca FS.
Mineral trioxide aggregate–based sealer: analysis of tissue reactions to a new endodontic material. J Endod 2010;36:1174–8.
5. Hasturk H, Kantarci A, Van Dyke TE. Oral inflammatory diseases and systemic inflammation: role of the macrophage. Front. Immun 2012;3:118.
6. Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol 2008;8:958-69.
7. Mosser DM. The many faces of macrophage activation. J Leukoc Biol 2003;73:209-12.
8. Mills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM. M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol 2000;164:6166–73.
36 10.Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 2004;25:677-86.
11.Rezende TM, Vargas DL, Cardoso FP, Sobrinho AP, Vieira LQ. Effect of
mineral trioxide aggregate on cytokine production by peritoneal macrophages.
Int Endod J 2005;38:896-903.
12.Nathan C, Shiloh MU. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Nati Acad Sci U S A 2000;97:8841–8.
13.de Oliveira Mendes ST, Ribeiro Sobrinho AP, de Carvalho AT, de Souza Côrtes MI, Vieira LQ. In vitro evaluation of the cytotoxicity of two root canal sealers on macrophage activity. J Endod 2003;29:95-9.
14.van de Loosdrecht AA, Nennie E, Ossenkoppele GJ, Beelen RH, Langenhuijsen MM. Cell mediated cytotoxicity against U 937 cells by human monocytes and macrophages in a modified colorimetric MTT assay. A methodological study. J Immunol Methods 1991;141:15-22.
15.Lee A, Whyte MK, Haslett C. Inhibition of apoptosis and prolongation of
neutrophil functional longevity by inflammatory mediators. J Leukoc Biol
1993;54:283-8.
16.Giaimis J, Lombard Y, Makaya-Kumba M, Fonteneau P, Poindron P. A new and simple method for studying the binding and ingestion steps in the phagocytosis of yeasts. J Immunol Methods 1992;154:185–93.
37 18.Al-Hiyasat AS, Tayyar M, Darmani H. Cytotoxicity evaluation of various resin
based root canal sealers. Int Endod J 2010;43:148–53.
19.Lópes-López J, Estrugo-Devesa A, Jané-Salas E, Segura-Egea JJ. Inferior alveolar nerve injury resulting from overextention of an endodontic sealer: non-surgical management using GABA analogue pregabalin. Int Endod J 2012;45: 98-104.
20.Orstavik D, Mjôr IA. Usage test of four endodontic sealers in Macaca
fascicularis monkeys. Oral Surg Oral Med Oral Pathol 1992;73:337–44.
21.de Oliveira Mendes ST, de Brito LC, Rezende TM, de Oliveira Reis R, Cardoso
FP, Vieira LQ, Ribeiro Sobrinho AP. A decrease in innate immune response to
infection in the presence of root canal sealers. Oral Surg Oral Med Oral Pathol
Oral Radiol Endod 2010;109:315-23.
22.Bin CV, Valera MC, Camargo SE, Rabelo SB, Silva GO, Balducci I, Camargo CH. Cytotoxicity and genotoxicity of root canal sealers based on mineral trioxide aggregate. J Endod 2012;38:495–500.
23.Scelza MZ, Linhares AB, da Silva LE, Granjeiro JM, Alves GG. A multiparametric assay to compare the cytotoxicity of endodontic sealers with primary human osteoblasts. Int Endod J 2012;45:12–8.
24.Silva EJ, Rosa TP, Herrera DR, Jacinto RC, Gomes BP, Zaia AA. Evaluation of Cytotoxicity and Physicochemical Properties of Calcium Silicate-based Endodontic Sealer MTA Fillapex. J Endod 2013;39:274-7.
38 26.Mahdi JG, Alkarrawi MA, Mahdi AJ, Bowen ID, Humam D. Calcium salicylate mediated apoptosis in human HT-1080 fibrosarcoma cells. Cell Prolif 2006;39:249–60.
27.Keiser K, Johnson CC, Tipton DA. Cytotoxicity of mineral trioxide aggregate using human periodontal ligament fibroblasts. J Endod 2000;26:288–91.
28.Saidon J, He J, Zhu Q, Safavi K, Spangberg LS. Cell and tissue reactions to mineral trioxide aggregate and Portland cement. Oral Surg, Oral Med, Oral Pathol, Oral Radiol Endod 2003;95:483–9.
29.Koh ET, McDonald F, Pitt Ford TR, Torabinejad M. Cellular response to mineral trioxide aggregate. J Endod 1998;24:543–7.
30.Mitchell PJ, Pitt Ford TR, Torabinejad M, McDonald F. Osteoblast biocompatibility of mineral trioxide aggregate. Biomaterials 1999;20:167–73. 31.Haglund R, He J, Jarvis J, Safavi KE, Spangberg LS, Zhu Q. Effects of root-end
filling materials on fibroblasts and macrophages in vitro. Oral Surg, Oral Med, Oral Pathol, Oral Radiol Endod 2003;95:739–45.
32.Unanue ER, Allen PM. The immunoregulatory role of the macrophage. Hosp
Pract (Off Ed) 1987;22:87-98.
33.Zhu Q, Haglund R, Safavi KE, Spangberg LS. Adhesion of human osteoblasts on root-end filling materials. J Endod 2000;26:404–6.
34.Sadeghein A, Bolhari B, Sarafnejad A. A comparison of the effect of three
endodontic sealers on adherence of mouse peritoneal macrophages. J Calif Dent
Assoc 2001;29:673-7.
39 36.Jiménez-Rubio A, Segura JJ, Llamas R, Jinénez-Plamas A, Guerrero JM, Calvo JR. In vitro study of the effect of sodium hypochlorite and glutaraldehyde on substrate adherence capacity of macrophages. J Endod 1997;23:562–4.
40 Figure Legends
Figure 1 – The percentage of living macrophages (a) from C57BL/6 mice and macrophages (b) from BALB/c mice, after incubation of the culture plates with capillaries containing MTA or MTA Fillapex. The controls were cultured with empty capillaries. The cultures were maintained for 24 h as described in Materials and Methods. Bars represent the mean of three experiments; lines represent the standard error of the means. (c) The percentage of living macrophages from C57BL/6 and from BALB/c treated with MTA Fillapex at different time-points. indicates a statistically significant difference between groups (p<0.05).
Figure 2 – The percentage of living macrophages (a) from C57BL/6 mice (a) and macrophages from BALB/c mice (b), after MTT assay with capillaries containing MTA or MTA Fillapex. The controls were cultured with empty capillaries. The cultures were maintained for 24 h as described in Materials and Methods. Bars represent the mean of three experiments; lines represent the standard error of the means. indicates a statistically significant difference between groups (p<0.05).
41
between groups (p<0.05). #, Ф and ∆ indicate significant differences between
macrophage sources (p<0.05).
42
Figure 1
* * 0 20 40 60 80 100 (a) % o f l iv in g m a c ro p h a g e s 0 20 40 60 80 100MTA MTA Fillapex Control
(b)
0 2 4 6 8 10 12
0 50 100 150 M1 M2 % o f li v in g m a c ro p h a g e s
43
Figure 2
*
0 20 40 60 80 100 % o f li v in g m a c ro p h a g e s (a) (b)*
*
*
*
*
MTA MTA Fillapex
44
45
Figure 4
M1 macrophages
M2 Macrophages
0
20
40
60
80
100
120
Control
MTA
MTA fillapex
46
The effect of a mineral trioxide aggregate- based sealer on the
production of reactive oxygen species, nitrogen species and cytokine by
two macrophage subtypes.
Julia Mourão Braga1, Ricardo Reis Oliveira1, Renata de Castro Martins2, Antonio
Paulino Ribeiro Sobrinho1.
1Departamento de Odontologia Restauradora, Faculdade de Odontologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil;
47 Abstract
Aim
To test the effects of a mineral trioxide aggregate-based sealer (MTA Fillapex®) and MTA (MTA-Ângelus®) on viability and on the production of cytokines, reactive oxygen species (ROS) and nitrogen species (NO) by M1 and M2 inflammatory macrophages.
Methodology
M1 (from C57BL/6 mice) and M2 (from BALB/c mice) peritoneal inflammatory macrophages were obtained and cultured in vitro in the presence of original and diluted extracts of MTA and MTA Fillapex (FLPX). The cellular viability, ROS production and the production of tumour necrosis factor-a, interleukin (IL)-12, IL-10 and NO in
response to stimulation with interferon-ɣ and Fusobacterium nucleatum or
Peptostreptococcus anaerobius were evaluated. The data were analysed using the
Mann–Whitney test and Student’s t-test.
Results
48
Conclusions
MTA treatment resulted in had excellent superior macrophage viability and cytokine production. FLPX impaired important effector responses to bacteria in both macrophage types.
49 Introduction
Successful endodontic therapy consists of cleaning and shaping procedures followed by tridimensional filling of the root canal with gutta-percha and sealer. Independent of the physicochemical properties of the sealers, if these materials are not biocompatible, it can cause degeneration of the periapical tissue and delay wound healing (Murray et al. 2007, Sousa et al. 2009). Recently, MTA Fillapex (Angelus, Londrina, PR, Brazil), an MTA-based sealer, was introduced to the market in an attempt to combine the physicochemical properties of a root canal sealer with the biological properties of Mineral trioxide aggregate (MTA) (Torabinejad & Chivian 1999, Rezende
et al. 2007, Scarparo et al. 2010).
Frequently, root canal sealers extrude or contact periapical tissues beyond the apical foramen, and these sealers can occasionally cause tissue inflammation. In this latter condition, the recruitment of inflammatory cells and the release of pro-inflammatory and immune regulatory cytokines have occurred (Stashenko et al., 1998, Brito et. al., 2011). Macrophages predominated among the cells that were recruited to this area (Van Dyke 2008, Hasturk et al. 2012). The main functions of macrophages include the elimination of invading bacteria, recruitment of other cells to the site of infection, clearance of excess neutrophils, production of cytokines and chemokines, and activation of the lymphocyte-mediated adaptive immune response (Taylor et al.2005, Hasturk et al. 2012).
50 concentrations of IL-10 and low levels of IL-12 (Mantovani et al. 2002, Bastos et al. 2002, Edwards et al. 2006, Verreck et al. 2004). Moreover, in pro-inflammatory M1 cells, high levels of inductive nitric oxide synthase (iNOS) result in citrulline and nitric oxide (NO) production. In M2 cells, arginine metabolism leads to ornithine and urea production, which culminates in collagen synthesis and cell proliferation (Mosser 2003, Bogdan et al. 1991, Bronte & Zanovello 2005).
51 Materials and Methods
Mice
Male and female C57BL/6 and BALB/c mice between 4 to 8 weeks of age were obtained from CEBIO (UFMG, Belo Horizonte, Brazil) and kept in a conventional animal house with barriers, temperature and light control. Food and water were offered ad libitum.
Isolation of macrophages
Cells were isolated from the peritoneal cavity of C57BL/6 (M1 macrophages) and Balb/c (M2 macrophage) mice 5 days after peritoneal injection of 2 mL of 3% thioglycolate medium (Biobrás S.A., Montes Claros, MG, Brazil). Cells were resuspended in complete medium: RPMI 1640 (Sigma Chemicals Co., St. Louis, MO, USA), supplemented with 10% foetal calf serum (Nutricell, Campinas, SP, Brazil), 0.1% of 0.05 mol L-1 β-mercaptoethanol (Sigma Chemicals Co.), 0.2% penicillin (100 U mL-1)/streptomycin (0.1 mg mL-1) and 200 mmol L-1 L-glutamine (Oliveira Mendes et al. 2003).
Preparation of Extracts
MTA and MTA Fillapex® (FLPX) were prepared in sterile conditions in accordance
52 original extracts (1:1) were then serially diluted (1:2, 1:4, 1:8, 1:16 and 1:32) in cell culture medium before testing.
Cytotoxicity Testing
The viability of cells in the presence of the original extracts and in serial dilutions was tested by culturing 1 x 106 cells in 96-well culture plates for 24 and 72 hours. One hundred microliters of RPMI 1640 supplemented with 10% foetal bovine serum, penicillin, and streptomycin containing 1 x 106 cells/well were seeded in 96-well plates and incubated for 24 hours at 37⁰C. The cells were then exposed to 200 µL of the original extracts and their serial dilutions. After 24 hours, cell survival was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO) (Van de Loosdrecht et al. 1991). The results were expressed as the percentage of viable cells. Experiments were reproduced in triplicate.
Microorganism preparation
To induce tumour necrosis factor (TNF), reactive oxygen species (ROS), IL-12, IL-10 and NO, root canal pathogens were selected: Fusobacterium nucleatum (ATCC 10953), a negative bacterium, and Peptostreptococcus anaerobius (ATCC 27337), a gram-positive bacterium. Zymosan A from Saccharomyces cerevisiae (Sigma Chemical Co., St. Louis, MO) was used as a positive control for the ROS assay (Rezende et al. 2007). The microorganisms were grown in brain heart infusion broth (BHI; Difco, Detroit, MI), supplemented and prereduced (BHI-PRAS) in an anaerobic chamber (Forma Scientific, Marietta, OH) containing an atmosphere composed of 85% N2, 10%
H2, and 5% CO2 for 48 hours at 37°C. The samples were adjusted in phosphate-buffered
53 methodology proposed by Ribeiro Sobrinho et al. (2002) was used. Zymosan A was diluted in PBS (109 particles/mL) (Trusk et al. 1978).
Reactive oxygen intermediates assay
Reactive oxygen species were assayed as described by Trusk et al. (1978)with slight modifications. Macrophages (1 x 106 cells/well) were transferred to a C96 White Maxisorp (Nalgene, Rochester, New York, USA) plate in a total volume of 200 µL that contained: 0.05 mmol/L luminol, diluted extract (1:4) and 107 zymosan particles (Sigma Chemical Co.) in RPMI 1640 without phenol red. Diluted extract was not used in control groups. The plates were read in a luminometer every 2 minutes for a total of 118 minutes (LumiCount Packard Instrument Company, Downers Grove, IL). The results were expressed as the area under each of the curves.
Cell culture and cytokine assays
54 Statistical analysis
The data were analysed using parametric (Student’s t-test) and nonparametric (Mann– Whitney) tests (P < 0.05). Analyses were made using the SPSS 18.0 Inc. (Statistical Package for Social Sciences, Chicago, IL, USA) software.
Results Cell Viability
Cell viability using MTT is demonstrated in Fig. 1. When the control group was compared to the test group, FLPX sealer was extremely cytotoxic at the highest concentrations (1:1, 1:2) and significantly decreased the viability of both macrophage types at 24 h and 72 h after stimulation (p < 0.05). However, cells treated with diluted FLPX extracts (1:4, 1:8) remained viable ( 80%) for 24 h and 72 h. (Fig. 1a, b). Macrophages of both subtypes remained viable (80%) after stimulation with diluted MTA extracts (1:1, 1:2, 1:4 and 1:8) (Fig. 1c, d). No differences were found between the viabilities of M1 and M2 macrophages.
ROS assay
Analysis of the area under each ROS production curve showed that zymosan stimulated significantly higher respiratory bursts for M2 macrophages compared to M1 macrophages (Fig. 2). Figure 2 demonstrates that FLPX sealer inhibited the production
55 NO production
M1 and M2 macrophages produced similar levels of NO in all of the conditions examined. The only exception was observed in Peptostreptococcus anaerobius-stimulated M2 macrophages, where high amounts of NO were observed (p≤0.046). NO production by M1 and M2 cells was increased when IFN-ɣ was added to the cultures (Fig. 3). The addition of Fusobacterium nucleatum-stimulated cells induced NO production, and these levels were higher when IFN-ɣ was added to the culture. MTA decreased NO production in M1 F.nucleatum-stimulated cultures with IFN-ɣ stimuli
(p≤0.001). As observed for ROS, FLPX sealer decreased NO production in F.
nucleatum and P. anaerobius -stimulated M1 and M2 macrophages (p<0.05).
TNF-α
M2 macrophages produced higher levels of TNF-α compared to M1 macrophages in all
of the conditions tested (p≤0.05) (Fig. 4). FLPX sealer induced TNF-α production in P. anaerobius-stimulated M1 macrophage cultures in the presence of IFN-ɣ (p≤0.05) (Fig. 5a). TNF-α production by F. nucleatum-stimulated M1 cells was not influenced by the
presence of FLPX sealer. However, FLPX sealer significantly decreased TNF-α production in the presence or absence of IFN-ɣ in F. nucleatum-stimulated M2 macrophages (p≤0.05) (Fig. 4b).
IL-12p70
IL-56 12p70 production by M1 macrophages in all conditions (p<0.05) except for F.
nucleatum-stimulated cultures (Fig. 5).
IL-10
Figure 4 shows that M2 macrophages produced higher levels of IL-10 than M1 macrophages in all conditions tested (p<0.05). FLPX sealer stimulated the production of IL-10 by M1 and M2 macrophages in P. anaerobius-stimulated macrophages cultured in the presence or absence of IFN-ɣ. MTA also stimulated IL-10 production by M2 macrophages in P. anaerobius and F. nucleatum -stimulated cultures (Fig. 6). M2 macrophages that were pre-incubated with MTA extract and subsequently treated with
F. nucleatum antigen in the absence of IFN-ɣ produced the highest levels of IL-10 (p<0.05) (Fig. 6b).
Discussion
Studies to assay the effects of root canal sealers on macrophage function bring important knowledge about the innate and adaptive immune responses in inflamed periapical tissues surrounding teeth subjected to endodontic treatments. Professional antigen-presenting cells (APCs) such as macrophages ingest foreign particles, including infectious agents and cellular debris (Kopitar et al. 2006, Hasturk et al. 2012). Several studies have shown that endodontic materials can impair the phagocytic activities of periapical APCs (Lee et al. 2007, Sousa et al. 2009, de Oliveira Mendes et al. 2010, Brackett et al. 2011).
57 response to sealers during the inflammation (M1 macrophages) and healing (M2 macrophages) processes.
The first step is to analyse the cell viability in the presence of sealers. As previously shown (Rezende et al.2005), MTA did not affect M1 or M2 macrophage viability. However, FLPX sealer significantly decreased the cell viability of both macrophage subtypes after stimulation with high extract concentrations (1:1, 1:2). Accordingly, Bin et al. (2012) observed similar results when using original and serially diluted extracts of these sealers. Several other groups have confirmed these findings (Scelza et al.2011, Silva et al. 2013, Salles 2012). It is possible that toxic components in FLPX, such as salicylate resin, could be responsible for the negative outcomes observed here (Stark et al.2001, Mahdi et al. 2006, Faria-Júnior et al. 2013). After analysing these findings, we determined that the best concentration of FLPX and MTA was 1 to 4 (1:4) because this ratio did not interfere with macrophage viability.
The secretory activity of stimulated macrophages was investigated using this sealer concentration. After being phagocytised, microorganisms are killed via ROS and NO production (Garcia & Stein 2006, de Oliveira Mendes et al.2010). Therefore, we tried to analyse whether sealers would interfere with this response in the presence or absence of endodontic pathogens or zymosan (yeast positive control for ROS).
58 previously observed for M1 macrophages treated with EWT Pulp Canal sealer (de Oliveira Mendes et al., 2010), even though others have shown that MTA treatment does not affect ROS production (Rezende et al., 2007; Camargo et al., 2009).
The FLPX sealer decreases NO production by M1 and M2 macrophages in F.
nucleatum and P. anaerobius-treated cells. Yoshino et al. (2013) reported low levels of
NO in fibroblast culture supernatants after FLPX extracts were added. In contrast, NO production by bacteria-treated M1 and M2 macrophages increased when IFN-ɣ was added to the cultures, as described elsewhere (Rezende et al.2007; de Oliveira Mendes
et al. 2010). As described previously, there was no difference in NO production with
MTA treatment when compared with the control group (Rezende et al.2007). Taken together, these results show that FLPX sealer impairs the immune response against endodontic pathogens and decreases NO and ROS production.
Pro-inflammatory mediators, such as cytokines, and microbial challenge, are responsible for the changes that occur in inflamed periapical tissues (Stashenko et al., 1998). Macrophages secrete a number of cytokines that perpetuate inflammation or direct healing processes (Sjogren et al. 1998, Brackett et al. 2011).Therefore, we were interested in whether FLPX sealer interferes with TNF-α, IL-12 and IL-10 production by both macrophage subtypes. These cytokines are involved in the onset of the inflammatory processes (TNF-α), in the interconnection of the innate and adaptive immune responses (IL-12), or in immune regulation (IL-10). Heat-killed bacteria and IFN-ɣ stimuli were added to cell cultures to reproduce clinical conditions because anaerobic bacteria and other cytokines are frequently found in compromised periapical tissues (Resende et al., 2005).
59 microorganisms (Locksley et al. 2001, Xanthoulea et al. 2004); it also indirectly induces osteoclast activation (Stashenko 1990, Wang et al., 1997, Maciel et al., 2011). In contrast to a previous study, M2 macrophages produced higher levels of TNF-α than M1 macrophages (Resende et al., 2005). FLPX sealer induced TFN-α production in P. anaerobius-stimulated M1 macrophages cultured in the presence of IFN-ɣ (p≤0.05).
However, TNF-α production by F. nucleatum-stimulated M1 cells was not influenced
by the presence of FLPX sealer. The opposite was observed in M2 cells: FLPX sealer significantly decreased TNF-α production in F. nucleatum-stimulated cells cultured in the presence or absence of IFN-ɣ. Other studies have also found that TNF-α production by macrophages was impaired by sealers (Perassi et al. 2004, Sousa et al. 2009, Brackett et al. 2009, de Oliveira Mendes et al. 2010). Moreover, MTA did not have negative effects on TNF-α production by M1 and M2 macrophages, as demonstrated by others (Rezende et al., 2005, 2007). These results suggest that in the absence of residual infection, MTA and FLPX sealers do not interfere with the production of TNF-α by either macrophage subtype.
60 in control cells and in P. anaerobius-stimulated cells, as demonstrated elsewhere (Resende et al., 2005).
The M2 profile is involved in the progression of infectious diseases as well as in the healing process (Benoit et al.; 2008; de Oliveira Mendes et al., 2010). Collagen synthesis and cell proliferation is associated with IL-10 production (Kawashima &
Stashenko, 1999). Interleukin-10 induces tissue homeostasis, which leads to the
inhibition of pro-inflammatory cytokine production by activated T cells and macrophages (Kawashima et al. 1996, Stashenko et al. 1998, Gerber & Mosser 2001, Borish & Steinke 2003, Brito et al., 2012). As expected, M2 macrophages produced higher levels of IL-10 than did M1 macrophages. FLPX sealer stimulated the production of IL-10 by M1 and M2 macrophages in P. anaerobius- stimulated cells cultured in the presence or absence of IFN-ɣ. Moreover, M2 macrophages that were pre-incubated with MTA extract and subsequently treated with F. nucleatum antigen in the absence of IFN-ɣ produced the highest levels of IL-10 (p<0.05) (Fig. 4b). Previously, it was demonstrated that M2 macrophages treated with F. nucleatum in the absence of the sealers produced higher levels of IL-10 than did M1 macrophages under the same conditions (de Oliveira Mendes et al., 2010).
61 Acknowledgements
62
References
Bastos KR, Alvarez JM, Marinho CR, Rizzo LV, Lima MR (2002) Macrophages from IL-12p40-deficient mice have a bias toward the M2 activation profile. Journal
of Leukocyte Biology 71, 271–8.
Benoit M, Desnues B, Mege JL (2008) Macrophage polarization in bacterial infections. Journal of Immunology 181, 3733-9.
Bin CV, Valera MC, Camargo SEA (2012) Cytotoxicity and genotoxicity of root canal sealers based on mineral trioxide aggregate. Journal of Endodontics 38, 495–500.
Bogdan C, Vodovotz Y, Nathan C (1991) Macrophage deactivation by interleukin 10. Journal of Experimental Medicine 174, 1549-55.
Borish LC, Steinke JW (2003) 2. Cytokines and chemokines. Journal
of Allergy and Clinical Immunology 111, 460-75.
Brackett MG, Marshall A, Lockwood PE, Lewis JB, Messer RLW, Bouillaguet S, Wataha JC (2009) Inflammatory Suppression by Endodontic Sealers After Aging 12 Weeks In Vitro. Journal of Biomedical Materials Research Part B Applied
63 Brackett MG, Lewis JB, Messer RL, Lei L, Lockwood PE, Wataha JC (2011)
Dysregulation of monocytic cytokine secretion by endodontic sealers. Journal of
Biomedical Materials Research Part B Applied Biomaterials 97, 49-57.
Bronte V, Zanovello P (2005) Regulation of immune responses by L-arginine metabolism. Nature Reviews Immunology 5, 641-54.
Camargo CH, Camargo SE, Valera MC, Hiller KA, Schmalz G, Schweikl H (2009) The induction of cytotoxicity, oxidative stress, and genotoxicity by root canal sealers in mammalian cells. Oral Surgery, Oral Medicine, Oral
Pathology, Oral Radiology, and Endodontology 108, 56-60.
de Oliveira Mendes ST, Ribeiro Sobrinho AP, de Carvalho AT, de Souza Cortes MI,
Vieira LQ (2003) In vitro evaluation of the cytotoxicity of two root canal sealers on
macrophage activity. Journal of Endodontics 29, 95-9.
de Oliveira Mendes ST, Brito LCN, Rezende TMB, Reis RO, Cardoso FP, Vieira
LQ,Ribeiro Sobrinho AP (2010) A decrease in innate immune response to infection
in the presence of root canal sealers. Oral Surgery, Oral Medicine, Oral
Pathology, Oral Radiology, and Endodontology 109, 315-323.
de Brito LC, Teles FR, Teles RP, Totola AH, Vieira LQ, Sobrinho AP (2012) T-lymphocyte and cytokine expression in human inflammatory periapical lesions.
64 Edwards JP, Zhang X, Frauwirth KA, Mosser DM (2006) Biochemical and
functional characterization of three activated macrophage populations. Journal of
Leukocyte Biology 80, 1298-307.
Faria-Júnior NB, Tanomaru-Filho M, Berbert FL, Guerreiro-Tanomaru JM (2013) Antibiofilm activity, pH and solubility of endodontic sealers. Journal of Endod
ontics 39, 274-7.
Garcia X, Stein F (2006) Nitric oxide. Seminars in pediatric infectious diseases 17, 55-7.
Gerber JS, Mosser DM. Reversing lipopolysaccharide toxicity by ligating the macrophage Fc gamma receptors (2001) Journal of Immunology 166, 6861-8.
Hasturk H, Kantarci A, Van Dyke TE (2012) Oral inflammatory diseases and systemic inflammation: role of the macrophage. Frontiers in Immunology 3,118.
Kawashima N, Kosaka T, Suda H (1996) Kinects of macrophages and lymphoid cells during development of experimentally induced periapical lesions in rat molars: a quantitative immunohistochemical study Journal of Endodontics 22, 311-326.
65 Kopitar-Jerala N (2006) The role of cystatins in cells of the immune system. FEBS
Letters 580, 6295-301.
Lee YY, Hung SL, Pai SF, Lee YH, Yang SF (2007) Eugenol suppressed the expression of lipopolysaccharide-induced proinflammatory mediators in human macrophages. Journal of Endodontics 33, 698-702.
Locksley RM, Killeen N, Lenardo MJ (2001) The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104, 487-501.
Maciel KF, Neves de Brito LC, Tavares WL, Moreira G, Nicoli JR, Vieira LQ, Ribeiro Sobrinho AP (2012) Cytokine expression in response to root canal infection in gnotobiotic mice. International Endodontic Journal 45, 354-62.
Mahdi JG, Alkarrawi MA, Mahdi AJ, Bowen ID, Humam D. Calcium
salicylatemediated apoptosis in human HT-1080 fibrosarcoma cells (2006) Cell
Proliferation 39, 249–60.
Mantovani A, Sozzani S, Locati M, Allavena P, Sica A (2002) Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends in Immunology 23, 549-55.
66 Mosser DM (2003) The many faces of macrophage activation. Journal of Leukocyte
Biology 73, 209-12.
Murray PE, Garcia-Godoy F, Hargreaves KM (2007) Regenerative endodontics: a review of current status and a call for action. Journal of Endodontics 33, 377-90.
Rezende TM, Vargas DL, Cardoso FP, Sobrinho AP, Vieira LQ (2005) Effect of
mineral trioxide aggregate on cytokine production by peritoneal macrophages.
International Endodontic Journal 38, 896-903.
Perassi FT, Filho IB, Berbert FL, Carlos IZ, de Toledo Leonardo R (2004) Secretion of tumor necrosis factor-alpha by mouse peritoneal macrophages in the presence of dental sealers, Sealapex and Endomethasone. Journal of Endodontics 30, 534–537.
Rezende TM, Vieira LQ, Cardoso FP, Oliveira RR, de Oliveira Mendes ST, Jorge
ML (2007) The effect of mineral trioxide aggregate on phagocytic activity and
production of reactive oxygen, nitrogen species and arginase activity by M1 and M2
macrophages. International Endodontic Journal 40, 603-11.
67 Scarparo RK, Haddad D, Acasigua GAX (2010) Mineral trioxide aggregate–based sealer: analysis of tissue reactions to a new endodontic material . Journal of
Endodontics 36, 1174–8.
Scelza MZ, Linhares AB, da Silva LE, Granjeiro JM, Alves GG (2012) A multiparametric assay to compare the cytotoxicity of endodontic sealers with primary human osteoblasts. International Endodontic Journal 45, 12–8.
Silva EJNL, Rosa TP, Herrera DR, Jacinto RC, Gomes BPFA, Zaia AA (2013) Evaluation of Cytotoxicity and Physicochemical Properties of Calcium Silicate-based Endodontic Sealer MTA Fillapex. Journal of Endodontics 39, 274-277.
Sjogren U, Ohlin A, Sundqvist G, Lerner UH (1998) Gutta-perchastimulated mouse macrophages release factors that activatethe bone resorptive system of mouse calvarial bone. European Journal of Oral Sciences 106, 872–881.
Sousa LR, Cavalcanti BN, Marques MM (2009) Effect of laser phototherapy on the release of TNF-alpha and MMP-1 by endodontic sealer-stimulated macrophages.
Photomedicine and laser surgery 27, 37-42.
Stark LA, Din FV, Zwacka RM, Dunlop MG (2001) Aspirin-induced activation of the NFkappaB signaling pathway: a novel mechanism for aspirin-mediated
68 Stashenko P (1990) Role of immune cytokines in the pathogenesis of periapical lesions. Endodontic Dental Traumatology 6, 89–96.
Stashenko P, Teles R, D'Souza R (1998) Periapical inflammatory responses and their modulation. Criticial Reviews in Oral Biology & Medicine 9, 498-521.
Taylor PR, Martinez-Pomares L, Stacey M, Lin H H, Brown G D, Gordon S (2005) Macrophage receptors and immune recognition. Annual Review of Immunology 23, 901–944.
Torabinejad M, Chivian N (1999) Clinical applications of mineral trioxide aggregate. Journal of Endodontics 25, 197–205.
Trinchieri G, Scott P (1995) Interleukin-12: a proinflammatory cytokine with immunoregulatory functions. Research in Immunology 146, 423-31.
Trusk MA, Wilson ME, Dyke KV (1978) The generation of chemiluminescence by phagocytic cells. In: Marlene A, Deluca, ed. Methods in Enzymology. London, UK: Academic Press, pp. 462-93.
van de Loosdrecht AA, Nennie E, Ossenkoppele GJ, Beelen RH, Langenhuijsen MM (1991) Cell mediated cytotoxicity against U 937 cells by human monocytes and macrophages in a modified colorimetric MTT assay. A methodological study.
69 Van Dyke TE (2008) The management of inflammation in periodontal disease.
Journal of Periodontology 79, 1601-8.
Verreck FA, De Boer T, Langenberg DM, Hoeve MA, Kramer M, Vaisberg E, Kastelein R, Kolk A, de Waal-Malefyt R, Ottenhoff TH (2004) Human IL-23- producing type 1 macrophages promote but IL-10-producing type 2 macrophages subvert immunity to (myco) bacteria. Proceedings of the National Academy of
Sciences 101, 4560-5.
Xanthoulea S, Pasparakis M, Kousteni S, Brakebusch C, Wallach D, Bauer J,
Lassmann H, Kollias G (2004) Tumor necrosis factor (TNF) receptor shedding controls thresholds of innate immune activation that balance opposing TNF functions in infectious and inflammatory diseases. Journal of Experimental
Medicine 200, 367-76.
Wang CY, Tani-Ishii N, Stashenko P (1997) Boneresorptive cytokine gene expression in periapical lesions in the rat. Oral Microbiology and Immunology 12, 65–71.
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Figure legends
Figure 1- Percentage of living M1 macrophages (a and c) from C57BL/6 mice and M2 macrophages (b and d) from BALB/c mice after exposure to extracts and organised according to type of dilution. Original extracts (1:1) were serially diluted in fresh medium as indicated. Cell cultures were exposed to original extracts and to serially
diluted extracts for 24 hours □ and for 72 hours ■. Columns represent the mean cell
viability expressed as percentages. The bars represent the means of 3 experiments
performed independently in triplicate. Black stars ( ) indicate significant differences
between treatment with original and diluted extracts.
Figure 2- Production of reactive oxygen intermediates (ROS) by M1 macrophages (a) from C57BL/6 mice and M2 macrophages (b) from BALB/c mice. Representative kinetics of ROS production by zymosan-stimulated and unstimulated M2 macrophages in the presence or absence of sealers are shown in (c). Cells were cultured with diluted extracts of MTA and FLPX sealer (1:4) and stimulated with zymosan as described in materials and methods. Bars represent the mean results of three experiments performed in triplicate; lines represent the standard error of the mean. indicates a statistically significant difference between groups (p<0.05). Black stars ( ) indicate significant differences between control and stimulated groups, and white circles (
ʘ
) indicate significant differences between macrophage sources (p<0.05).71 of MTA and FLPX (1:4) and stimulated with F. nucleatum (F) or P. anaerobius (P), and IFN-gamma (I) as indicated. Bars represent the mean results of three experiments
performed in triplicate; lines represent the standard error of the mean. indicates a statistically significant difference between groups (p<0.05). Black stars ( ) indicate significant differences between control and stimulated groups, and white circles (
ʘ
) indicate significant differences between macrophage sources (p<0.05).Figure 4- Production of TNF by M1 (a) and M2 (b) macrophages cultured in the
absence (control) or presence of sealer extracts (1:4) 24 hours after incubation. The cells were cultured in the medium alone (M - control), or with Fusobacterium nucleatum (F) or Peptostreptococcus anaerobius (P) preparations. Interferon-ɣ (I) was added where indicated at 10 U/mL (1 µg = 8,430 U). The bars represent the means of 2 experiments performed independently in quadruplicate, and the lines represent the standard deviation of the means. indicates a statistically significant difference between groups
(p<0.05). Black stars (
) indicate significant differences between control and
stimulated groups, and white circles (ʘ
) indicate significant differences between
macrophage sources (p<0.05).Figure 5- Production of IL-12 by M1 (a) macrophages cultured in the absence or presence of diluted sealer extracts (1:4) 24 hours after incubation. The cells were cultured in medium alone (M - control), or with Fusobacterium nucleatum (F) or
72 of the means. M2 macrophages did not produce IL-12. Moreover, indicates a statistically significant difference between groups (p<0.05). Black stars ( ) indicate significant differences between control and stimulated groups (p<0.05).
Figure 6- Production of IL-10 by M1 (a) and M2 (b) macrophages cultured in the absence (control) or presence of diluted sealer extracts (1:4) 72 hours after incubation. The cells were cultured in medium alone (M - control) or with Fusobacterium
